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Microprobe analysis and fine structure of mitochondrial granules in ultrathin frozen sections of rat pancreas
Printed in Sweden Copyright © 1977 by Academic Press, Inc. All rights of reproduction in any form reserved ISSN 0014-4827
PRELIMINARY NOTES
Microprobe analysis and fine structure of mitochondrial granules in ultrathin frozen sections of rat pancreas 1 W. J. MERGNER, S. H. CHANG, R. T. JONES, and B. F. TRUMP, Department of Pathology, Univer-
sity of Maryland School of Medicine, Baltimore, MD 21201, USA Summary. High resolution electron microscopy and microprobe analysis studies on untreated frozen sections of normal pancreas reveal a distinct appearance of mitochondrial granules. These granules have been shown to contain a significant amount of calcium and potassium. Based upon these findings, it is proposed that normal mitochondrial granules represent special sites for cation storage and release since these structures are quite labile in contrast to the abnormal accumulation of divalent cations in loading experiments.
Precise information on ion concentration in normal and abnormal cells has not been accessible until recently when new methods of ultrathin frozen sectioning of rapidly frozen tissues and microprobe analysis of these frozen sections became available. Mitochondrial calcium content plays a crucial role in normal cells. Changes in the calcium concentration of the cytosol stimulates cellular functions such as muscle contraction or response to hormones. If calcium metabolism is disturbed, as it occurs in cell injury, then these functions are also disturbed. Little is known, however, about the active sites of calcium metabolism. Normal mitochondria contain granules which appear in glutaraldehyde-osmium tetroxide fixed and Epon-embedded sections as electron-dense, round bodies of 300-400 A diameter within the matrix space [1]. Based upon circumstantial evidence Peachy [2] postulated that these granules 1 This is publication no. 268 of the Cellular Pathobiology Laboratory and was supported by NIH grant AM 15440-4. 28-771802
are sites of divalent ion accumulation. Trump and co-workers [3] observed that mitochondrial granules quickly disappear following ischemic cell injury in the mouse liver in vitro. Densities of a quite different morphology are observed if isolated mitochondria are suspended in calcium (or another cation) containing solutions particularly in the presence of substrate, inorganic phosphate and/or ATP [4-9]. Densities which appear then are either (a) punctate calcium phosphate precipitates close to the membranes of cristae; or (b) microcrystals in the same location. After ashing, they have a crystal pattern of hydroxyapatite [8, 9]. These densities are also seen in certain types of cell injury [5, 6]. Their differences are characterized by their location in the matrix space and morphology. Previous studies using liquid fixatives had serious drawbacks because of the observed lability of mitochondrial granules [10]. Pasquali-Ronchetti et al. [11] disputed that calcium was a major component of normal dense granules because, in the presence of 2,4-dinitrophenol, 80% of mitochondrial calcium was released within 2 min while no decrease was observed in dense granules in the matrix. The granules were observed in mitochondria that had undergone routine fixation, dehydration, and embedding. It could be assumed, therefore, that during routine processing for electron microscopy, calcium disappears partially or totally and is replaced by osmium, uranyl, or lead at the binding sites [10, 12]. Christensen's [13] findings that mitochondrial granules (-600 A in diameter) are more abundant and pleomorphic in ultracryomicrotomy sections than in routine sections lends further credence to this assumption. Exp CellRes 108 (1977)
430
Preliminary notes
j 6 ~4 --o~ ~K z-co 9×le,~ls 3 , 7 e KEY ~e sEc OS" 256 AIS M HI" |J |U 8
;. ,, ,..,[',. ........ ,,,,i,,lllllllll,,i,il, Fig. 1. Normal pancreas, glutaraldehyde--osmium tetroxide fixation. ×30000. Fig. 2. Ultracryomicrotome section of rat pancreas. Note the contour of mitochondria with multiple electron-dense granules. × 30 000. Fig. 3. High resolution electron micrograph of a single Exp Cell Res 108 (1977)
granule, with inset to reveal ultrastructural arrangement of granule. Inset, × 300 000. Fig. 4. Microprobe analysis demonstrating a comparison between mitochondria matrix (bars) and granules (dots). Line is on Ca Kc~.
Preliminary notes Somlyo and his co-workers [14] demonstrated significant quantities of calcium that were associated with phosphorus in mouse heart mitochondria by electron probe analysis of unstained frozen thin sections. However, no mention was made of mitochondrial granules.
Materials, Methods and Results Pancreatic tissues from Sprague-Dawley rats were prepared for high resolution transmission electron microscopy and electron probe analysis by contact freezing in liquid nitrogen. Specimens were sectioned in an ultramicrotome fitted with a Cryokit [13] at -7&C. Sections were brought to room temperature in a desiccation chamber. High resolution studies were carried out on a JEOL 100B electron microscope operated at 60-80 kV. The microscope was fitted with a high resolution specimen holder, a 150 condenser and a 50/zm objective aperture. Microprobe analyses were conducted on both a JEOL JXA-50A scanning electron microscope fitted with an Ortec 165 eV energy dispersive analyzer with a Li drifted Si detector and a JEOL JEM 100C transmission microscope fitted with a KEVEX System 5100 X-ray energy spectrometer and a 185 eV energy resolution multichannel analyzer energy dispersive system fitted through the pole piece. Standards were frozen sections of serial concentrations of albumin--CaC12 solutions. Tissues fixed in glutaraldehyde followed by osmium tetroxide show a few small dense granules in the mitochondrial matrix space (fig. 1), while frozen tissues have numerous pleomorphic granules among the rough endoplasmic reticulum (fig. 2). These later granules have an elaborate substructure, which by high magnification, consists of an electron-dense matrix of horseshoe or ring-shaped spheres with electron-translucent tings (fig. 3). Microprobe analysis of these granules demonstrated calcium and potassium peaks (fig. 4). The calcium peaks had both Ka and Kfl lines while the mitochondrial matrix distant from granules had no calcium peak and a lower potassium peak. There was no difference in the height of the phosphate peak between cytoplasm, mitochondrial matrix or granules (not shown in fig. 4).
Discussion Our studies demonstrate that mitochondrial granules have an elaborate substructure and they contain calcium. The granules may represent the site of calcium sequestration by mitochondria and once calcium is accumulated it apparently does not remain in an ionic form but becomes bound in an amor-
431
phous pattern. This suggests a mechanism to carry out the functions of calcium sequestration [15]. Other studies have documented an elaborate and effective release mechanism of normally sequestered mitochondrial calcium in response to sodium [16] in contrast to massive calcium loading where amorphous crystalline or semicrystalline patterns are observed and calcium is not readily released [5, 6]. The substructure of the mitochondrial granules that we have observed corresponds to the non-crystalline precipitation seen in early stages of extracellular calcification [17, 18]. Based on their ultrastructure, we propose that the granules consist of calcium and other cations in an amorphous arrangement possibly but not necessarily, in combination with phosphate, since phosphate levels were not particularly elevated. The finding that localized sites were responsible for ion accumulation in mitochondria points out a fascinating aspect of metabolism which requires further investigation. We feel presently that our data do not support the notion that calcium in granules is necessarily present in the form of calcium phosphate salts, since the phosphate peak was of equal height whether or not granules and/or calcium were pre'sent in mitochondria. Also the relation of these granules to those seen after the usual type of liquid fixation is not known. An alternative possibility has not been excluded by our studies. This alternative mechanism could explain the occurrence of mitochondrial granules as the result of rapid local concentration and possibly precipitation of electrolytes secondary to extraction of water during the freezing process. Calcium would then normally be sequestered in some other form than in granules. The elevated peak of potassium over the elecExp Cell Res 108 (1977)
432
Preliminary notes
t r o n - d e n s e granules could f a v o r this p o s sibility. The assistance of D. Harling of JEOL Corporation Applications Laboratory, Medford, MA, is greatly appreciated. References
1. Palade, G E, Anat rec 114 (1952) 427. 2. Peachy, L D, J cell biol 20 (1964) 95. 3. Trump, B F, Goldblatt, P J & Stowell, R E, Lab invest 14 (1965) 343. 4. Greenawalt, J W, Rossi, C S & Lehninger, A L, J cell biol 23 (1966) 21. 5. Sahaphong, S, Dissertation, Duke University, Durham, NC (1971). 6. Sahaphong, S & Trump, B F, Amj patho163 (1971) 277, 7. Lehninger, A L, Biochemj 119 (1970) 129. 8. Thomas, R S & Greenawalt, J W, J cell biol 39 (1968) 55. 9. Weinbach, E C & Von Brand, T, Biochim biophys acta 148 (1967) 256. 10. Fawcett, D W, The cell, its organelles and inclusions, p. 79. Saunders, Philadelphia, Pa (1966). 11. Pasquali-Ronchetti, I, Greenawalt, J W & Carafoli, E J, J cell bio140 (1969) 565. 12. Sutfin, L, Holtrop, M E & Ogilvie, R E, Science 174 (1971) 947. 13. Christensen, K J, J cell biol 51 (1971) 772. 14. Somlyo, A P, Somlyo, A V, Devine, C E, Peters, P D & Hall, T A, J cell biol 61 (1974) 723. 15. Lehninger, A, The mitochondrion: Molecular basis of structure and function. Benjamin, New York (1965). 16. Carafoli, E, Tiozzo, R, Lugli, G, Crovetti, F & Kratzing, C, J mol cell cardiol 6 (1974) 361. 17. Weber, J C, Eanes, E P & Gerdes, R J, Arch biochem biophys 120 (1974) 723. 18. Molner, J, J ultrastruct res 3 (1959) 39. Received December 20, 1976 Revised version received May 9, 1977 Accepted May 31, 1977
nuclei isolated from rat skeletal muscles. Significantly higher levels of activity of RNA polymerase B were found in the nuclei isolated from the slow-twitch soleus compared with nuclei from the fast-twitch extensor digitorum longus. A l t h o u g h the difference b e t w e e n slowtwitch and fast-twitch skeletal m u s c l e s with r e s p e c t to their protein c o m p o s i t i o n a n d t u r n o v e r is well r e c o g n i z e d [1, 2], the role p l a y e d b y the m o t o r n e u r o n in the regulation o f p r o t e i n synthesis in its c o n t i g u o u s m u s c l e cell is not u n d e r s t o o d . I f n e u r o t r o phic influences are exerted at the level o f gene e x p r e s s i o n in m u s c l e [2--4], then o n e r e g u l a t o r y site w h i c h requires investigation is the rate o f gene transcription b y the R N A p o l y m e r a s e s . T h e lack o f i n f o r m a t i o n on n u c l e a r R N A synthesis in m a m m a l i a n skeletal m u s c u l a t u r e is p r o b a b l y related to the difficulties o f obtaining g o o d yields o f pure nuclei f r o m this fibrous tissue [5]. I n this r e p o r t , the optimal i n c u b a t i o n conditions f o r e v a l u a t i o n o f the o~-amanitin-resistant and ot-amanitin-sensitive D N A - d e p e n d e n t R N A p o l y m e r a s e s (nucleoside triphosphate : RNA nucleotidyltransferase, E C 2.7.7.6) in nuclei isolated f r o m t w o different t y p e s o f skeletal muscle, i.e., the red, slow-twitch soleus and the white, fasttwitch e x t e n s o r digitorum longus o f the r a t are c o m p a r e d . Materials and Methods
Evaluation of R N A polymerase activities in nuclei isolated from slow and fast skeletal muscles IRENE R. HELD, Neurosciences Research Labora,o0', Veterans Administration Hospital, Hines, 1L 60141, and Loyola University Stritch School of Medicine, Maywood, IL 60153, USA Summary. The optimal incubation conditions were
determined for the assay of the ~-amanitin-resistant, DNA-dependent RNA polymerase A and the a-amanitin-sensitive, DNA-dependent RNA polymerase B in Exp CelIRes 108 (1977)
The sodium salts of ATP, CTP (type I) and GTP (type I), pyruvate kinase (PK), phosphoenolpyruvate (PEP), cysteine, pancreatic deoxyribonuclease I, actinomycin D, a-amanitin, RNA (type III) and DNA (type I) were obtained from Sigma Chemical Co., St Louis, Mo. The [4-14C]UTP, ammonium salt (60 /zCi/ttmole) in 2% ethanol from Amersham/Searle Corp., Des Plaines, I11., was lyophilized and dissolved in deionized water shortly before use. All of the inorganic chemicals were obtained from Fisher Scientific Co., Pittsburgh, Pa. The soleus and extensor digitorum longus (EDL) muscles were excised from the hind limbs of male, albino, Wistar rats, 3-6 months of age, within several minutes after decapitation and placed immediately in ice-cold homogenizing media (0.32 M sucrose, 3 mM MgC12, pH 6.8). The distinct procedures that
PRELIMINARY NOTES
Microprobe analysis and fine structure of mitochondrial granules in ultrathin frozen sections of rat pancreas 1 W. J. MERGNER, S. H. CHANG, R. T. JONES, and B. F. TRUMP, Department of Pathology, Univer-
sity of Maryland School of Medicine, Baltimore, MD 21201, USA Summary. High resolution electron microscopy and microprobe analysis studies on untreated frozen sections of normal pancreas reveal a distinct appearance of mitochondrial granules. These granules have been shown to contain a significant amount of calcium and potassium. Based upon these findings, it is proposed that normal mitochondrial granules represent special sites for cation storage and release since these structures are quite labile in contrast to the abnormal accumulation of divalent cations in loading experiments.
Precise information on ion concentration in normal and abnormal cells has not been accessible until recently when new methods of ultrathin frozen sectioning of rapidly frozen tissues and microprobe analysis of these frozen sections became available. Mitochondrial calcium content plays a crucial role in normal cells. Changes in the calcium concentration of the cytosol stimulates cellular functions such as muscle contraction or response to hormones. If calcium metabolism is disturbed, as it occurs in cell injury, then these functions are also disturbed. Little is known, however, about the active sites of calcium metabolism. Normal mitochondria contain granules which appear in glutaraldehyde-osmium tetroxide fixed and Epon-embedded sections as electron-dense, round bodies of 300-400 A diameter within the matrix space [1]. Based upon circumstantial evidence Peachy [2] postulated that these granules 1 This is publication no. 268 of the Cellular Pathobiology Laboratory and was supported by NIH grant AM 15440-4. 28-771802
are sites of divalent ion accumulation. Trump and co-workers [3] observed that mitochondrial granules quickly disappear following ischemic cell injury in the mouse liver in vitro. Densities of a quite different morphology are observed if isolated mitochondria are suspended in calcium (or another cation) containing solutions particularly in the presence of substrate, inorganic phosphate and/or ATP [4-9]. Densities which appear then are either (a) punctate calcium phosphate precipitates close to the membranes of cristae; or (b) microcrystals in the same location. After ashing, they have a crystal pattern of hydroxyapatite [8, 9]. These densities are also seen in certain types of cell injury [5, 6]. Their differences are characterized by their location in the matrix space and morphology. Previous studies using liquid fixatives had serious drawbacks because of the observed lability of mitochondrial granules [10]. Pasquali-Ronchetti et al. [11] disputed that calcium was a major component of normal dense granules because, in the presence of 2,4-dinitrophenol, 80% of mitochondrial calcium was released within 2 min while no decrease was observed in dense granules in the matrix. The granules were observed in mitochondria that had undergone routine fixation, dehydration, and embedding. It could be assumed, therefore, that during routine processing for electron microscopy, calcium disappears partially or totally and is replaced by osmium, uranyl, or lead at the binding sites [10, 12]. Christensen's [13] findings that mitochondrial granules (-600 A in diameter) are more abundant and pleomorphic in ultracryomicrotomy sections than in routine sections lends further credence to this assumption. Exp CellRes 108 (1977)
430
Preliminary notes
j 6 ~4 --o~ ~K z-co 9×le,~ls 3 , 7 e KEY ~e sEc OS" 256 AIS M HI" |J |U 8
;. ,, ,..,[',. ........ ,,,,i,,lllllllll,,i,il, Fig. 1. Normal pancreas, glutaraldehyde--osmium tetroxide fixation. ×30000. Fig. 2. Ultracryomicrotome section of rat pancreas. Note the contour of mitochondria with multiple electron-dense granules. × 30 000. Fig. 3. High resolution electron micrograph of a single Exp Cell Res 108 (1977)
granule, with inset to reveal ultrastructural arrangement of granule. Inset, × 300 000. Fig. 4. Microprobe analysis demonstrating a comparison between mitochondria matrix (bars) and granules (dots). Line is on Ca Kc~.
Preliminary notes Somlyo and his co-workers [14] demonstrated significant quantities of calcium that were associated with phosphorus in mouse heart mitochondria by electron probe analysis of unstained frozen thin sections. However, no mention was made of mitochondrial granules.
Materials, Methods and Results Pancreatic tissues from Sprague-Dawley rats were prepared for high resolution transmission electron microscopy and electron probe analysis by contact freezing in liquid nitrogen. Specimens were sectioned in an ultramicrotome fitted with a Cryokit [13] at -7&C. Sections were brought to room temperature in a desiccation chamber. High resolution studies were carried out on a JEOL 100B electron microscope operated at 60-80 kV. The microscope was fitted with a high resolution specimen holder, a 150 condenser and a 50/zm objective aperture. Microprobe analyses were conducted on both a JEOL JXA-50A scanning electron microscope fitted with an Ortec 165 eV energy dispersive analyzer with a Li drifted Si detector and a JEOL JEM 100C transmission microscope fitted with a KEVEX System 5100 X-ray energy spectrometer and a 185 eV energy resolution multichannel analyzer energy dispersive system fitted through the pole piece. Standards were frozen sections of serial concentrations of albumin--CaC12 solutions. Tissues fixed in glutaraldehyde followed by osmium tetroxide show a few small dense granules in the mitochondrial matrix space (fig. 1), while frozen tissues have numerous pleomorphic granules among the rough endoplasmic reticulum (fig. 2). These later granules have an elaborate substructure, which by high magnification, consists of an electron-dense matrix of horseshoe or ring-shaped spheres with electron-translucent tings (fig. 3). Microprobe analysis of these granules demonstrated calcium and potassium peaks (fig. 4). The calcium peaks had both Ka and Kfl lines while the mitochondrial matrix distant from granules had no calcium peak and a lower potassium peak. There was no difference in the height of the phosphate peak between cytoplasm, mitochondrial matrix or granules (not shown in fig. 4).
Discussion Our studies demonstrate that mitochondrial granules have an elaborate substructure and they contain calcium. The granules may represent the site of calcium sequestration by mitochondria and once calcium is accumulated it apparently does not remain in an ionic form but becomes bound in an amor-
431
phous pattern. This suggests a mechanism to carry out the functions of calcium sequestration [15]. Other studies have documented an elaborate and effective release mechanism of normally sequestered mitochondrial calcium in response to sodium [16] in contrast to massive calcium loading where amorphous crystalline or semicrystalline patterns are observed and calcium is not readily released [5, 6]. The substructure of the mitochondrial granules that we have observed corresponds to the non-crystalline precipitation seen in early stages of extracellular calcification [17, 18]. Based on their ultrastructure, we propose that the granules consist of calcium and other cations in an amorphous arrangement possibly but not necessarily, in combination with phosphate, since phosphate levels were not particularly elevated. The finding that localized sites were responsible for ion accumulation in mitochondria points out a fascinating aspect of metabolism which requires further investigation. We feel presently that our data do not support the notion that calcium in granules is necessarily present in the form of calcium phosphate salts, since the phosphate peak was of equal height whether or not granules and/or calcium were pre'sent in mitochondria. Also the relation of these granules to those seen after the usual type of liquid fixation is not known. An alternative possibility has not been excluded by our studies. This alternative mechanism could explain the occurrence of mitochondrial granules as the result of rapid local concentration and possibly precipitation of electrolytes secondary to extraction of water during the freezing process. Calcium would then normally be sequestered in some other form than in granules. The elevated peak of potassium over the elecExp Cell Res 108 (1977)
432
Preliminary notes
t r o n - d e n s e granules could f a v o r this p o s sibility. The assistance of D. Harling of JEOL Corporation Applications Laboratory, Medford, MA, is greatly appreciated. References
1. Palade, G E, Anat rec 114 (1952) 427. 2. Peachy, L D, J cell biol 20 (1964) 95. 3. Trump, B F, Goldblatt, P J & Stowell, R E, Lab invest 14 (1965) 343. 4. Greenawalt, J W, Rossi, C S & Lehninger, A L, J cell biol 23 (1966) 21. 5. Sahaphong, S, Dissertation, Duke University, Durham, NC (1971). 6. Sahaphong, S & Trump, B F, Amj patho163 (1971) 277, 7. Lehninger, A L, Biochemj 119 (1970) 129. 8. Thomas, R S & Greenawalt, J W, J cell biol 39 (1968) 55. 9. Weinbach, E C & Von Brand, T, Biochim biophys acta 148 (1967) 256. 10. Fawcett, D W, The cell, its organelles and inclusions, p. 79. Saunders, Philadelphia, Pa (1966). 11. Pasquali-Ronchetti, I, Greenawalt, J W & Carafoli, E J, J cell bio140 (1969) 565. 12. Sutfin, L, Holtrop, M E & Ogilvie, R E, Science 174 (1971) 947. 13. Christensen, K J, J cell biol 51 (1971) 772. 14. Somlyo, A P, Somlyo, A V, Devine, C E, Peters, P D & Hall, T A, J cell biol 61 (1974) 723. 15. Lehninger, A, The mitochondrion: Molecular basis of structure and function. Benjamin, New York (1965). 16. Carafoli, E, Tiozzo, R, Lugli, G, Crovetti, F & Kratzing, C, J mol cell cardiol 6 (1974) 361. 17. Weber, J C, Eanes, E P & Gerdes, R J, Arch biochem biophys 120 (1974) 723. 18. Molner, J, J ultrastruct res 3 (1959) 39. Received December 20, 1976 Revised version received May 9, 1977 Accepted May 31, 1977
nuclei isolated from rat skeletal muscles. Significantly higher levels of activity of RNA polymerase B were found in the nuclei isolated from the slow-twitch soleus compared with nuclei from the fast-twitch extensor digitorum longus. A l t h o u g h the difference b e t w e e n slowtwitch and fast-twitch skeletal m u s c l e s with r e s p e c t to their protein c o m p o s i t i o n a n d t u r n o v e r is well r e c o g n i z e d [1, 2], the role p l a y e d b y the m o t o r n e u r o n in the regulation o f p r o t e i n synthesis in its c o n t i g u o u s m u s c l e cell is not u n d e r s t o o d . I f n e u r o t r o phic influences are exerted at the level o f gene e x p r e s s i o n in m u s c l e [2--4], then o n e r e g u l a t o r y site w h i c h requires investigation is the rate o f gene transcription b y the R N A p o l y m e r a s e s . T h e lack o f i n f o r m a t i o n on n u c l e a r R N A synthesis in m a m m a l i a n skeletal m u s c u l a t u r e is p r o b a b l y related to the difficulties o f obtaining g o o d yields o f pure nuclei f r o m this fibrous tissue [5]. I n this r e p o r t , the optimal i n c u b a t i o n conditions f o r e v a l u a t i o n o f the o~-amanitin-resistant and ot-amanitin-sensitive D N A - d e p e n d e n t R N A p o l y m e r a s e s (nucleoside triphosphate : RNA nucleotidyltransferase, E C 2.7.7.6) in nuclei isolated f r o m t w o different t y p e s o f skeletal muscle, i.e., the red, slow-twitch soleus and the white, fasttwitch e x t e n s o r digitorum longus o f the r a t are c o m p a r e d . Materials and Methods
Evaluation of R N A polymerase activities in nuclei isolated from slow and fast skeletal muscles IRENE R. HELD, Neurosciences Research Labora,o0', Veterans Administration Hospital, Hines, 1L 60141, and Loyola University Stritch School of Medicine, Maywood, IL 60153, USA Summary. The optimal incubation conditions were
determined for the assay of the ~-amanitin-resistant, DNA-dependent RNA polymerase A and the a-amanitin-sensitive, DNA-dependent RNA polymerase B in Exp CelIRes 108 (1977)
The sodium salts of ATP, CTP (type I) and GTP (type I), pyruvate kinase (PK), phosphoenolpyruvate (PEP), cysteine, pancreatic deoxyribonuclease I, actinomycin D, a-amanitin, RNA (type III) and DNA (type I) were obtained from Sigma Chemical Co., St Louis, Mo. The [4-14C]UTP, ammonium salt (60 /zCi/ttmole) in 2% ethanol from Amersham/Searle Corp., Des Plaines, I11., was lyophilized and dissolved in deionized water shortly before use. All of the inorganic chemicals were obtained from Fisher Scientific Co., Pittsburgh, Pa. The soleus and extensor digitorum longus (EDL) muscles were excised from the hind limbs of male, albino, Wistar rats, 3-6 months of age, within several minutes after decapitation and placed immediately in ice-cold homogenizing media (0.32 M sucrose, 3 mM MgC12, pH 6.8). The distinct procedures that